The present invention relates to an apparatus for measuring minute membrane potential, which measures a difference in potential between areas separated by a membrane or different positions on a membrane, such as an interior and exterior of a cell membrane, as well as an electrode constituting this apparatus.
To deal with an aging society, a technical research area for directly supporting human lives is being expanded. This is called a xe2x80x9cbarrier-free technologyxe2x80x9d which supports deteriorated or lost functions of human bodies by improving the physical structures of social infrastructures such as towns and houses or developing new personally available equipment. Technical developments based on this concept contribute to creating an environment where human beings live easy social lives whether or not they are physically handicapped.
The barrier-free technology initially concentrated on electronics, machinery, and construction but is being gradually expanded; some recent studies for the barrier-free technology are directly related to medicine. Typical examples include research on advanced medical equipment such as functional artificial hands or legs which supplement the functions of the bodies of handicapped people or the like. Such equipment is now controlled using methods such as electromyographic detection or detection of motions of the eyes or tongue, but information obtained by these input means is quantitatively significantly insufficient in terms of the amount of information required for motor and sensory functions of living bodies. If, for example, a person has lost his hand or leg due to a traffic accident, or a war wound, he must have an artificial hand or leg. However, to control the operation of the artificial hand or leg as if it were an actual hand or leg and to feed back senses for temperature or contacts to the human body, a certain interface is required which can simultaneously measure action potential generated by individual nerves of a bundle of several hundred peripheral nerves and then input a corresponding signal. Already developed such interfaces, however, are all insufficient.
One of the most successful examples in the field of direct exchange of information between nerves and electronic equipment is equipment called artificial inner ear [Rehabilitation Medicine, 31, 233-239 (1994)]. If an auditory defect occurs such that a portion between the tympanic membrane and the inner ear is prevented from functioning, the artificial inner ear executes the function of this portion by directly stimulating auditory nerves in the cochlear organ. The artificial inner ear processes a sound input through a microphone depending on the auditory characteristic of this person to generate a digital signal in order to electrically stimulate terminals of the auditory nerves, that is, cochlear nerves. Spherical electrodes are generally arranged at about 22 locations inside the cochlea, where about 30,000 auditory nerves are located, to input a sound signal to the nerves through the electrodes. For some time after the artificial inner ear has been implanted, most subjects feel that they hear very strange sounds, but once information processing in the brain has been adapted to the artificial inner ear, the subjects can distinguish sounds. Analysis and improvements of electrodes for the artificial inner ear is a large research area.
A substantial reduction in input auditory information from an inherent input through 30,000 nerves to an artificial input through about 22 electrodes means that auditory information received by the brain also decreases down to 0.1% or less. Thus, regeneration of the sound listening ability depends on whether the brain can supplement and understand a substantially reduced amount of information. Several months are required to optimally adapt the auditory processing function of the brain to the artificial inner ear. The artificial inner ear suggests possibilities of nerve interfaces.
With the artificial inner ear, however, the electrodes are installed in the cochlear organ, where nerve terminals are exposed and lie over several centimeters. The nerve stimulating electrodes may be installed at nerve terminals exposed to the interior of the organ, so that it is not physically difficult to arrange the electrodes. In addition, in inputting a sound signal, it is very easy to analyze and determine those of the nerves which receive the signal and the frequency of the signal that can be received by these nerves. In this manner, the reasons why the artificial inner ear has successfully been put to practical use include many appropriate conditions for transmission of signals to the nerves through the electrodes. In other words, it is virtually impossible to directly apply the electrode technology for the artificial inner ear to nerves other than the cochlear nerves.
Many research institutes in the world are developing interfaces for exchanging information between nerve cells and electronic equipment; these studies can be roughly classified into two aspects.
One of them is a medical technological approach where minute electrodes are implanted in a nerve bundle or the brain [IEEE Trans. Biomed. Eng., 39, 893-902 (1992)] [IEEE Trans. Biomed. Eng. 41, 567-577 (1994)] [IEEE Trans. Biomed. Eng., 41, 305-313 (1994)]. This approach attempts to provide terminals for obtaining a control signal for rehabilitation equipment directly from nerves or multichannel connection terminals for inputting a signal to nerves.
The other aspect is based on a long-term prospect for application of the information processing ability of nerve cells to computers [Bioelectrochemistry and Bioenergetics, 29, 193-204 (1992)] [Brain Research, 446, 189-194 (1988)]. This approach pursues the possibilities of what is called xe2x80x9cbiocomputersxe2x80x9d that use living cells as operation elements.
Conventional nerve interfaces are roughly classified into the following three types:
(a) Aggregate needle-shaped metal electrodes and needle point holder-shaped aggregate metal needle electrodes [IEEE Trans. Biomed. Eng., 41, 1136-1146 (1994)]
(b) Axon regenerated matrix electrodes [IEEE Trans. Biomed. Eng., 39, 893-902 (1992)] [IEEE Trans. Biomed. Eng., 41, 567-577 (1994)] [IEEE Trans. Biomed. End., 41, 305-313 (1994)]
Flat cultured-nerve-cell electrodes on a substrate [Bioelectrochemistry and Bioenergetics, 29, 193-204 (1992)] [Brain Research, 446, 189-194]
Needle-shaped electrodes for measuring nerves have been used since the initial period of research on nerves, but the study of microneurography was the first to record the action potential of a single human peripheral nerve in situ [Clinical Electroencephalogram, 25, 493-500, 564-571, 629-638 (1983)]. The needle point holder-shaped electrode is one of the aggregate types in which this electrode is formed at a tip of a needle or on a side thereof and which is struck to a severed nerve bundle or a tissue in the brain [IEEE Trans. Biomed. Eng., 41, 1136-1146 (1994)]. That is, the needle point holder-shaped electrode is obtained by three-dimensionally expanding the needle-shaped electrode using a micromachine technology. In a basic design, the needle-shaped electrode records a faint extracellular current from a nerve that is accidentally located close to the electrode section. Although the needle-shaped electrode can be used to measure a single nerve cell, this method is evidently not accurate enough to simultaneously measure a large number of nerves even if the degree of integration of needle-shaped electrodes is increased to enhance spatial resolution, because the relative distance between each nerve and the electrode depends on accidents.
The axon regenerated matrix electrode in 2 is a field that has been expanded since 1992 when Stanford University conducted a relevant study, and many reports have recently been made on this electrode [IEEE Trans. Biomed. Eng. 39, 893-902 (1992)] [IEEE Trans. Biomed. Eng., 41, 567-577 (1994)] [IEEE Trans. Biomed. ENg., 41 305-313 (1994)]. This electrode is obtained by integrating electrodes into a 16xc3x9716 matrix or the like with a hole formed in each electrode section. The electrode is sandwiched between severed portions of a nerve bundle to record an extracellular current from each nerve axon regenerated through the corresponding hole in the electrode. The axon regenerated electrode is advantageous in that the axon and the electrode are physically stably joined together and that a signal from the nerve axon passed through the hole in the electrode can be identified and detected. However, severing the nerve bundle obviously adversely affects the nerves, and the nerve axons regenerated through the holes in the electrodes amounts to only several percents of the entire nerve bundle. The axon regenerated electrode is expected to have its degree of integration increased so as to detect current in each nerve to electrically stimulate each nerve. If, however, attempts are made to increase the degree of integration, the area of the electrode section per nerve must be reduced while the area required for a wiring section of the electrode must be increased. Consequently, the rate of opening necessarily declines to significantly affect the spatial resolution.
The flat electrode method in 3 is often used for basic studies for biocomputers [Bioelectrochemistry and Bioenergetics, 29, 193-204 (1992)] [Brain Research, 446, 189-194 (1988)]. In terms of extension of axons of nerve cells, various useful information has been obtained on the shape and material of a substrate to which a cell or an axon adheres and on an applied voltage. A problem of this method, however, is a small contact area between the electrode and the cell. A sufficient voltage and a sufficient contact area between the electrode and the cell are required to generate an action potential from the cell. Application of a high voltage, however, results in electrolysis of moisture, which is essential for a cell environment when the voltage exceeds about 1.3 V. Thus, the applicable voltage is limited. Since current density is also limited, the contact area between the cell and the electrode must necessarily be increased in order to effectively stimulate the cell. As a result, it is impossible to stimulate each single nerve cell, and the only possible method is to stimulate a mass of nerve cells on the electrode to obtain an integration effect.
A common disadvantage of these existing nerve interference is that they use the extracellular electrode to measure an electromotive force of about several xcexcv induced by an action potential in the nerve based on a minor change in the concentration of ions outside the cell membrane. First, when the spatial resolution of the electrode is increased, the electrode picks up extracellular currents from cells in the neighborhood. Second, when the nerve is electrically stimulated to input a signal thereto, the electrode must provide a current much higher than an extracellular current from the nerve, thereby preventing measurements by other electrodes in the neighborhood.
The essence of nerve information is a membrane potential pulse from the nerve cell having a variation of about 100 mV. Accordingly, appropriate electrodes for use in exchanging information between the nerve and the equipment must be able to contact hard with the cell membrane or to be inserted through the cell membrane in order to measure variations in membrane potential.
The above-described interfaces using the extracellular electrodes can obtain a certain amount of information from the nerve and stimulate the nerve. None of these electrodes, however, has an enough spatial resolution to join to each nerve axon on a one-to-one correspondence, the nerve axon constituting a fiber for transmitting information. This is essentially disadvantageous in mutually separating nerve signals. Except for the central and terminal portions, the entire nerve axon has a mixture of inputs from a sensory organ to the center (afferent nerve fibers) and outputs from the center to a muscle or the like (efferent nerve fibers). Thus, when these interfaces have a low spatial resolution, input signals from the sensory organ and output signals to the muscle or the like are likely to be crossed. When nerve signals obtained are resolved and separated into signals for the individual nerves, a problem occurs even with the matrix electrode, which appears to have been most successful among the interfaces and which have been experimented in the situ systems.
That is, in controlling such human body supporting equipment, the biggest problem to be solved is how to efficiently exchange information directly between a normal nerve system and the electronic equipment. Thus, various research institutes are studying xe2x80x9cnerve interfacesxe2x80x9d using electrodes that can each be connected directly to a nerve, which is a source of living information, in order to transmit information between the nerve and various equipment.
As described above, almost all the disadvantages of the existing nerve interfaces are related to the use of the extracellular electrodes. The current electrodes that can input and output information to and from the individual nerve cell through the cell membrane include minute electrodes and patch lamp electrodes both used for electrophysicological experiments. The problems listed below, however, must be solved before these methods can be used for actual applications.
1) Although a tip portion of the electrode which contacts with the cell is thin and has a diameter between 200 nm and 2 xcexcm, it is essential to integrate these electrodes together because their main body is composed of a glass tube of diameter several millimeters.
2) When the electrode is connected to the cell, it may destroy the cell membrane unless vibration of a measuring system is minimized.
3) Since an operation of connecting the electrodes to the cell is difficult, skills are required for the connection operation.
To insert an existing electrode into the cell, a physical shearing force must be applied to the electrode to destroy the cell membrane. If the electrode uses as a support, a material of a high physical strength such as glass, the cell membrane can be destroyed by applying a physical shearing force to the electrode. This method, however, is inappropriate for minute metal electrodes in terms of structure and strength.
The existing cell membrane destroying method using only a physical shearing force is not the best method because the membrane is shaped for a high fluidity. That is, a sharp electrode alone cannot always penetrate the cell membrane. It is particularly difficult to insert the electrode into a cell in the order of several tens of micrometers simply by physically pressing the electrode into the cell. A major reason why the patch electrode [Nature, 260, 799-802 (1976)][Pflugers Arch., 391, 85-100 (1981)] has been designed is that even the minute glass electrode, which is relatively strong, has the above problem; it is thus important to improve the electrode inserting method.
Some reports on the measurement of surfaces of living nerve cells using atomic force microscope show that the cells were not damaged to the degree that the cytoplasm was subjected to leakage despite an increase in a contact pressure of a probe for scanning and measuring a surface shape [Journal of Microscopy (Oxford), 182, 114-120 (1996)]. This is because the cell membrane is composed of phospholipid, which has a high fluidity. This high fluidity was clarified through studies of cell fusion at the end of the 1970s [Proc. Natl. Acad. Sci. 69, 2056-2060 (1972)][J. Am.Chem.Soc., 94, 4475-4481 (1972)] [Biochem.Med., 15, 212-216 (1976)].
It is thus contemplated that temporary and partial destruction of the cell membrane is used to assist the insertion of the electrode. Such a cell membrane destroying method requires a destroyed portion and the amount of destruction to be controlled. Enzymatic destruction using lipase or protease or a method using xcex2-rays or laser beams are possible, but the inventors have focused on a phospholipid radical chain peroxidation reaction as an example of destruction of the cell membrane using a method other than the physical shearing force.
Activated oxygen such as singlet oxygen or superoxide radicals peroxidizes unsaturated phospholipid in the cell membrane through a chain reaction. In contrast, the cell has an oxidation defending mechanism such as xcex1-tocopherol (vitamin E), which is an agent for capturing radicals in the membrane), or L-ascorbic acid (vitamin C) or superoxide dismutase (SOD), which is a water-soluble antioxidant, to resist oxidation. Defects in cells originating from radicals and the detailed mechanism of oxidation and defense there against are described in the document [xe2x80x9cFree Radicals in Biology and Medicinexe2x80x9d, Oxford University Press (1985)].
When such a chain oxidation action exceeds the oxidation defensing capability, the destruction of the phospholipid membrane progresses rapidly and exponentionally to deprive an ion penetration inhibiting capability of the cell membrane, thereby disabling the cell from maintaining metabolism.
Molecules that generate activated oxygen to trigger this lipid chain peroxidation reaction when irradiated with light are called xe2x80x9cphotosensitizers (PS)xe2x80x9d. General photosensitizers include rose bengal and porphyrin.
By using such a photosensitizer to modify the electrode so that it can act as a membrane destroyer to allow the electrode to penetrate the cell membrane, the cain peroxidization reaction has only to be effected on a minimum area of a target cell surface for a short period of time. Moreover, if the membrane is damaged due to the peroxidation reaction during an electrode inserting operation, it is expected to be repaired by the above-described antioxidation system after the insertion.
It is a basic object of the present invention to develop a technology for controlling the destruction of the cell membrane. For applications the simply require the cell to be destroyed, various poisons have been examined for a long time. No technology has met the cell technology""s need to partially and temporarily destroy the cell membrane without causing cell death. In addition, the physical-shearing-force-based method using minute glass tubes or the like is limited. That is, it is an object of the present invention to develop a technology for using a method other than the physical shearing force to punch a living membrane while controlling the destruction of the membrane, that is, a membrane destruction controlling technology, thereby developing an electrode that can denature or destroy the membrane and that can be put to practical use.
To punch the living membrane while controlling the destruction thereof, a destroyed portion and the amount of destruction must be controlled. Thus, the inventors have developed a method for enabling the membrane to be denatured or punched while controlling activation of membrane destruction.
Although it is possible to temporarily and partially denature/destroy the membrane using enzymatic destruction with lipase or protease or using xcex2-rays or laser beams, the inventors have focused on a phospholipid radical chain peroxidization reaction as an example of destruction of the cell membrane using a method other than the physical shearing force.
Activated oxygen such as singlet oxygen or superoxide radicals peroxidizes unsaturated phospholipid in the cell membrane through a chain reaction. In contrast, the cell has an oxidation defending mechanism such as xcex2-tocopherol (vitamin E), which is an agent for capturing radicals in the membrane), or L-ascorbic acid (vitamin C) or superoxide dismutase (SOD), which is a water-soluble antioxidant, to resist oxidation.
When such a chain oxidation action exceeds the oxidation defensing capability, the destruction of the phospholipid membrane progresses rapidly and exponentially to deprive an ion penetration inhibiting capability of the cell membrane, thereby disabling the cell from maintaining metabolism. As this chain membrane destruction progresses, the cell is finally killed.
Molecules that generate activated oxygen a trigger this lipid chain peroxidization reaction when irradiated with light are called xe2x80x9cphotosensitizers (PS)xe2x80x9d. General photosensitizers include rose bengal and porphyrin.
By using such a photosensitizer as a membrane denaturant, the chain peroxidization reaction has only to be effected partly on a target cell surface, that is, in a minimum area thereof for a short period of time, in order to denature the membrane. Moreover, if the membrane is damaged due to the peroxidization reaction during an electrode punching operation, it is expected to be repaired due to the fluidity of the membrane itself or by the above-described antioxidation system after the punching.
The inventors applied 5xe2x80x25xe2x80x3-bis(aminomethyl)-2,2,xe2x80x2:5xe2x80x22xe2x80x3-terthiophene dihydrochloride (BAT), one of the photosensitizers, to surface membranes of cultured cells PC12 from the nerve system. The photosensitizer is a membrane denaturant that can be controlled by means of light irradiation. Membrane resistance was measured to determine that the photosensitizer, as activated when the entire cell is irradiated with light, raises the membrane resistance, that is, ion permeability of the membrane. The inventors have also clarified that the amounts of light and photosensitizer can be controlled to control a change in membrane resistance caused by light irradiation, to three levels.
1) No effect
2) Recovery after a decrease in resistance
3) Loss of resistance
Furthermore, the inventors have found a feature of this method that the ion permeability of the membrane recovers to its state prior to destruction in about 30 seconds under preferred conditions.
A similar change in membrane resistance was observed when only the axon of the cell was irradiated with laser beams.
Furthermore, to determine whether the denaturing of the cell membrane using the photosensitizer is applicable to introduction of a substance to the cell, the inventors attempted to apply it to a microinjection process. For the microinjection process, an injection liquid containing the water-soluble fluorescent dyeing reagent Lucifer Yellow CH (LY) was prepared and whether or not the LY could be injected into the PC12 cell was used as an index for determining whether or not the injection was successful. In addition, an electric manipulator was used to automate the injection process to minimize the artificial effects on evaluation of the success rate.
With this injection process system, measurements were made to determine how the injection success rate varied depending on the presence of 100 xcexcM of the photosensitive 5xe2x80x25xe2x80x3-bis(aminomethyl)-2,2xe2x80x2:5xe2x80x22xe2x80x3-terthiophene dihydrochloride in the injection liquid or the presence of a process for irradiating the membrane with light from a 100-W mercury lamp.
As a result, when the injection liquid containing the photosensitizer was used and the light irradiation was executed, the injection success rate was about 80%. In other control examples, the rate was about 0 to 10%. Therefore, membrane denaturing has been confirmed to significantly improve the injection success rate.
Furthermore, using a rate at which the cell retained the LY after the injection process, as an index for a cell survival rate, the cell survival rate was compared between the photosensitive process and the normal process. Photosensitized cells exhibited a survival rate of about 90% for three to six days, which is significantly higher than that of normally processed cells, that is, about 10%.
Thus, the combination of the photosensitizer with light has been shown to serve to appropriately punch the membrane. That is, conditions for repairing the membrane without killing the cells can be easily determined depending on the level of membrane destruction. Of course, it is easy to produce a membrane destroying member with a membrane destroyer such as the photosensitizer applied to a support and then bring the membrane destroying member in contact with the membrane.
The inventors further produced a new member by providing a scanning probe of an atomic force microscope with an electrode and applying 5xe2x80x25xe2x80x3-bis(aminomethyl)-2,2xe2x80x2;5xe2x80x22xe2x80x3-terthiophene dihydrochloride to a probe section. When the member with this photosensitizer applied thereto is inserted into the cell membrane, resistance originating from the cell membrane is observed between the electrodes located inside and outside the membrane. Since a physical shearing force applied by the electrode of the atomic force microscope is not strong enough to punch the cell, it has been shown that the newly produced member can be used as one acting as the electrodes of the atomic force microscope and providing a controllable membrane destroying function.
Next, the inventors attempted to achieved a spatial resolution of 20 xcexcm using as an representative example of a nerve interface, one having such a specific target size of its own that it can be applied to mammalian peripheral nerves. The value of 20 xcexcm was set because peripheral myelinated nerves (A fibers) that carry out normal muscle control and sense transmission have a diameter between 1 and 22 xcexcm.
The inventors further selected a rate phechromocyte PC12 cell as a nerve model. The PC12 cell was established by Greene and Tischler in 1976 [Proc. Natl. Acad. Sci. USA 73, 2434-2428 (1976)]. This call is characterized to differentiate into a cell similar to a nerve when a nerve growth factor (NGF) is added thereto and is commonly used as a nerve cell model.
To examine a cell membrane penetrating nerve interface according to the present invention, physical conditions such as insertion speed and pressure which are required to insert minute metal electrodes into the cell must be evaluated. As an electrode system that could quantitatively examine these conditions, a scanning probe of an atomic force microscope (AFM) was provided with an electrode and used with the AFM. This system has the following advantages:
1) An image of the cell can be picked up to specify an electrode inserting position.
2) The electrode can be manipulated at a spatial resolution in the order of nanometers.
3) Since the system operates under digital control, electrode connection conditions can be quantitatively optimized.
4) Attempts can be made to automatically connect the cell and the electrode together by controlling the positions of the electrode based on program operations.
5) A probe section of a commercially available ATM probe is shaped like a rectangular pyramid of base and height each 10 xcexcm, so that the target spatial resolution of the 20 xcexcm can be sufficient achieved even with modifications required to provide the probe with electrode.
Data obtained by the AFM probe electrode can be used to develop a nerve interface using a membrane-penetrating metal electrode.
This enables membrane potential to be measured by bringing the electrode into close contact with the membrane. To measure a potential in the cell, however, the electrode must be inserted into the cell using a certain method. The conventional method uses a physical shearing force to insert a glass capillary or the like. A major problem with this method, however, is that if the minute electrode is composed of metal, it is support has an insufficient physical strength to penetrate the membrane.
The inventors have thus designed and developed a method for applying a cell membrane destroying technology using a method other than the physical shearing force, to insertion of the electrodes. By way of example, the inventors have succeeded in allowing the electrode to easily penetrate the membrane by coupling an electrode inserting operation to a cell membrane destroying operation using a compound that generates an activated oxygen species.
By way of a specific example, the inventors succeeded in producing an atomic force microscope actually equipped with a cell membrane destroying probe electrode, selecting an electrode installing portion while measuring the shape of a surface of the cell, and then inserting the electrode into the cell membrane. Those skilled in the art can integrate a number of electrodes together as appropriate using the above single electrode as a prototype.
That is, the present invention includes:
(1) an electrode comprising an insulated support, a conductive pattern formed on a surface of the insulated support, an insulator formed on the conductive pattern in such a manner that a portion on the conductive pattern which comes in contact with at least a membrane after the membrane has been penetrated can be insulated, and a membrane denaturation reaction prompting portion formed in the portion coming in contact with the membrane or in a neighborhood thereof and having a membrane denaturing force other than a physical shearing force,
(2) the electrode according to (1), wherein the insulated support comprises a particular support and an insulating layer covering a surface of the support,
(3) the electrode according to (1) or (2), wherein a compound that causes the membrane denaturation reaction prompting portion to induce membrane denaturation reaction is applied or fixed to the electrode,
(4) the electrode according to (3), wherein the membrane denaturation reaction utilizes a chained peroxidation reaction of membrane components started by a direct or indirect generation reaction of an activated oxygen species,
(5) the electrode according to (3) or (4), wherein the membrane denaturation reaction includes a reaction induced by a particular stimulus and a reaction precursor to denature or destroy the membrane,
(6) the electrode according to (5), wherein the particular stimulus is light and the reaction precursor is a photosensitizing compound,
(7) an electrode according to any one of (1), (2), and (3) to (6), wherein after the membrane has been denatured or destroyed, the electrode penetrates the membrane and the penetrated membrane comes in close contact only with part of the insulating portion,
(8) an electrode including a support comprising silicon processed by means of etching, wherein gold (Au) with a thickness of 220 nm is plated on a bottom surface (a measuring surface) of the support, areas of the electrode other than a measuring metal terminal and an equipment-connected metal terminal are insulated and covered by silicon dioxide with a thickness of 100 nm, and 5xe2x80x25xe2x80x3-bis(aminomethyl)-2,2xe2x80x2:5xe2x80x22xe2x80x3-terthiophene is applied to an electrode section,
(9) the electrode according to any one of (1) to (8), wherein the electrode is connected to a position controlling device, and a position where the electrode is inserted into or contacted with the membrane can be controlled,
(10) the electrode according to any one of (1) to (9), wherein the electrode is connected to the position controlling device and has a function for measuring a shape of a surface of the membrane or a solid,
(11) the electrode according to any one of (1) to (10), wherein the insulated support comprises a scanning probe of a scanning probe microscope,
(12) the electrode according to any one of (1) to (11), wherein at least a part of the measuring metal terminal is covered with an insulator membrane or a conductor membrane, and
(13) an interface type minute membrane potential measuring apparatus comprising the electrode and the potential measuring device according to one or more of (1) to (12).
The electrode provided by the present invention comprises the membrane denaturation reaction promoting portion having a membrane denaturing force other than the physical shearing force. Preferably, the electrode is connected to the position controlling device and, more preferably, it contacts therewith at such an arbitrary pressure that a surface of the membrane is not destroyed.
When the electrode is based on a common one including a support, a measuring metal terminal, an insulating portion and can retain electrode functions, it can be composed of common materials. For example, the support may preferably composed of silicon, glass, or the like. The material of the conductive pattern may be gold (Au), platinum (Pt), or other metal generally used for patterning in the field of electronics. An example of the insulator used in the insulating portion includes silicon dioxide or silicon nitride. The electrode may have additional functional, for example, an electrode position indicating function based on a fluorescent coating or an extended lifetime resulting from coating with an antiseptic unless the electrode functions are degraded.
The membrane denaturing portion of the electrode having the membrane denaturation reaction promoting portion refers to a portion that comes in contact with a particular portion of the membrane which is denatured or destroyed. The membrane denaturation reaction may be a chemical reaction including a reaction using radiation, laser beams, or the like, an enzymatic reaction using lipase or protease, or a chained peroxidization reaction of membrane components started by a direct or indirect generation reaction of an activated oxygen species. An example of the chained peroxidization reaction includes a lipid peroxidization reaction of a living membrane, and, more specifically, includes generation of a radical, singlet oxygen, superoxide, or generation of hydrogen peroxide.
Although the electrode does not necessarily require a substance causing the membrane denaturation reaction to be applied or fixed to the membrane denaturation reaction prompting portion if radiation or the like is used, it may have a compound applied or fixed to a portion thereof, the compound including an enzyme or a compound that involves generation of activated oxygen and causing the membrane denaturation reaction. Preferably, the applied or fixed compound is a reaction precursor that causes the membrane denaturation reaction when subjected to a particular stimulus. An example of the combination of the xe2x80x9cparticular stimulusxe2x80x9d and the xe2x80x9creaction precursorxe2x80x9d includes xe2x80x9clightxe2x80x9d and a xe2x80x9cphotosensitizerxe2x80x9dfor a light-induced electron transfer reaction, xe2x80x9cradiationxe2x80x9d and a xe2x80x9ccell membrane and moisture moleculesxe2x80x9d for a radiative chemical reactionxe2x80x9d, or a xe2x80x9cchange in electrode potentialxe2x80x9d and a xe2x80x9cconductive polymerxe2x80x9d for an electrode reaction.
More specifically, the photosensitizer that promotes generation of singlet oxygen when irradiated with light includes methylene blue, rose bengal, chlorophyll (a common name), hematoporphyrin, posoralen, bilirubin, riboflavin, chlorophyll, or retinal, and the compound that generates activated oxygen on the electrode upon application of a potential includes methylviologen. An example of the conductive polymer that is radicalized on the electrode when provided with a potential includes polyacetylene or polythiophen. A thiophen trimer (terthienyl) is a natural photosensitizer obtained from a plant and can be used as a compound that can induce membrane destruction upon application of light or a potential. The compound that catalyzes radicalization when irradiated with light may be semiconductor particles such as a ruhtenium (II) trisbipyridine complex or titanium oxide, or a starting agent that starts radical polymerization when irradiated with light may be used as a photosensitizer. For example, the photopolymerization sensitizer based on irradiation with ultraviolet rays may be a peroxide such as a benzoyl peroxide, an azo compound such as azobisisobutyronitryl, a carbonyl sulfur compound such as diacetyl, dibenzyl, etc. diphenyl monosulfide, diphenyl disulfide, dibenzoyl monosulfide, or dibenzoyl disulfide, a halogen compound such as CCl4, and a metallic salt such as FeCl3.
If the level of membrane destruction based on lipid peroxidizing cannot be easily controlled based only on the quantitative ratio of the xe2x80x9cparticular stimulusxe2x80x9d and the xe2x80x9ccompound activated by a stimulusxe2x80x9d or the like, the level of destruction may be controlled by adding a substance such as lipid or an oxidation inhibiting substance that weakens the lipid peroxidization reaction or assists membrane repairs.
Depending on the level of membrane denaturation, the electrode having the membrane denaturation reaction promoting portion as described above may contact with the membrane so as not to completely destroy it or may be used in such a manner that after the membrane has been denatured or destroyed, the electrode penetrates the membrane, which comes in close contact only with part of the insulating portion.
A specific example is an electrode including a support comprising silicon processed by means of etching, wherein gold (Au) with a thickness of 220 nm is plated on a bottom surface (a measuring surface) of the support, areas of the electrode other than a measuring metal terminal and an equipment-connected metal terminal are insulated and covered by silicon dioxide with a thickness of 100 nm, and 5xe2x80x25xe2x80x3-bis(aminomethyl)-2,2xe2x80x2:5xe2x80x22xe2x80x3-terthiophene is applied to an electrode section.
For the former type of electrode, that is, the electrode having the membrane denaturation reaction promoting portion, and the latter type of electrode, that is, the electrode connected to the position controlling device and which can contact with the surface of the membrane at such an arbitrary pressure that the membrane is not destroyed, both types of electrodes may be connected to the position controlling device to control the position where the electrode is inserted into or contacted with the membrane or may have a function for measuring the shape of the surface of the membrane or a solid, or may be a scanning probe of the scanning probe microscope.
In addition, both electrode can preferably be used even if at least part of the measuring metal terminal is covered with an insulated or genetic film, unless the electrode functions are lost. Further, an interface type minute membrane potential measuring apparatus can be provided which includes a combination of a plurality of the above-described electrodes and a potential measuring device. This interface type minute membrane potential measuring apparatus can also be provided as a composite interface type minute membrane potential measuring apparatus including an additional normal electrode or normal metal terminal, or a combination with another potential measuring terminal.
Alternatively, all the above-described electrodes or potential measuring devices can be used as nerve interfaces having the electrode connected to the individual nerve to transmit information between electric information equipment and the nerve, so that they can each be provided as part of precision equipment for use in research such as basic analysis and research on the brain, clarification of living cell mechanisms, or analysis of functional electric stimuli. Each of them can also be used as part of a highly integrated and very accurate invasive measuring type medical electrode or part of a connection and control device for living function substituting and supporting equipment such as an artificial organ. Any of the above-described electrodes or potential measuring devices can alternatively be used for part of a connection and control device for an artificial hand or leg comprising a joint or a sensory organ that can be controlled as in human bodies, or as part of a connection and control device between an artificial sensory organ (a visual or auditory sense) and a living body. Any of the above-described electrodes or potential measuring devices can alternatively be used as a part of a brain function expanding apparatus for patients who have their brains damaged due to the Alzheimer""s disease or the Parkinson""s disease or as a part of a living function expanding apparatus for recovering a living function lost due to a congenital disease. Communication among living things may be extended by connecting the above-described interface type minute membrane potential measuring apparatus using the electrode not only to human beings but also to general animals or even plants to clarify living information processing mechanisms.